Unmodified Histone H3K4 and DNA-dependent Protein Kinase Recruit Autoimmune Regulator to Target Genes

Autoimmune regulator (AIRE) directs the expression of otherwise tissue-restricted antigens (TRAs) in medullary thymic epithelial cells, allowing their presentation to developing T cells, which leads to central tolerance. We addressed the conundrum of how AIRE is recruited to these otherwise silent genes in cells. Our studies confirmed that interactions between AIRE and the unmodified histone H3K4 (H3K4me0) are important for targeting AIRE to the mouse insulin promoter in chromatin. By replacing its H3K4me0-binding module with one that binds to the methylated H3K4me3, we redirected the mutant AIRE.ING protein to an actively transcribed gene. Nevertheless, the mutant AIRE D297A protein, which could not bind to H3K4me0, still activated the human insulin promoter on an episomal plasmid target. This targeting was due to DNA-dependent protein kinase (DNA-PK). Thus, in cells that lacked the catalytic subunit of DNA-PK (DNA-PKcs), the assembly and activity of AIRE on DNA, whether in chromatin or on episomal plasmids, was abrogated. However, by the heterologous tethering of AIRE to DNA, we could restore its activity on a plasmid target in DNA-PKcs-negative cells. Importantly, mutations in the putative DNA-binding residues in its SAND domain had no effect on the transcriptional effects of AIRE. Thus, AIRE is recruited to TRA genes in chromatin via cooperative interactions with H3K4me0 and DNA-PK.